蛋白修饰定量质谱法研究两种不同作用模式基因毒化合物对组蛋白H3乙酰化的影响

引用本文: 瞿敏敏, 陈佳, 徐斌, 李治, 郭磊, 徐华, 谢剑炜. 蛋白修饰定量质谱法研究两种不同作用模式基因毒化合物对组蛋白H3乙酰化的影响[J]. 分析化学, 2022, 50(6): 940-947. doi: 10.19756/j.issn.0253-3820.221025 shu

Citation: QU Min-Min, CHEN Jia, XU Bin, LI Zhi, GUO Lei, XU Hua, XIE Jian-Wei. Determination of Genotoxic Compounds with Different Modes of Action on Histone H3 Acetylation by Protein Modification Quantitative Mass Spectrometry[J]. Chinese Journal of Analytical Chemistry, 2022, 50(6): 940-947. doi: 10.19756/j.issn.0253-3820.221025 shu

蛋白修饰定量质谱法研究两种不同作用模式基因毒化合物对组蛋白H3乙酰化的影响

军事科学院军事医学研究院毒物药物研究所, 抗毒药物与毒理学国家重点实验室, 北京 100850

通讯作者: 徐华,E-mail:huarxu@163.com

基金项目:
国家自然科学基金项目（No.21974151）资助。

摘要: 建立了细胞中组蛋白H3的9位（H3K9ac）或14位（H3K14ac）赖氨酸乙酰化及其相应原型特征多肽片段的高效液相色谱-三重四极杆质谱同时定量分析方法。样品经胰酶消化、丙酰化衍生、除盐及肽段富集等前处理后，在ACQUITY UPLCBEH C₁₈色谱柱上分离，采用质谱技术在正离子多反应监测模式（MRM）下检测。空白细胞样品中组蛋白H3的各特征肽段在1~250 ng/mL浓度范围内线性关系良好（R²> 0.99），检出限（LOD，S/N=3）均为0.1 ng/mL，准确度、精密度、基质效应及提取回收率均符合方法学要求。本方法具有操作简便、灵敏度高、检测速度快及定量分析准确等优势。将本方法应用于2种典型的不同作用模式的基因毒性化合物（喜树碱和秋水仙碱）对2种人源细胞株即肝癌细胞HepG2和宫颈癌细胞HeLa中组蛋白H3的9位或14位赖氨酸乙酰化修饰影响的研究，测得了组蛋白H3中H3K9ac及H3K14ac修饰含量的变化，探讨了其与不同基因毒性化合物作用模式的关系，为基因毒性化合物的DNA损伤/修复及转录研究提供了技术支持。

关键词:

English

Determination of Genotoxic Compounds with Different Modes of Action on Histone H3 Acetylation by Protein Modification Quantitative Mass Spectrometry

State Key Laboratory of Toxicology and Medical Countermeasures, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

Corresponding author: XU Hua, huarxu@163.com

Received Date: 14 January 2022
Revised Date: 31 March 2022
Fund Project:
Supported by the National Natural Science Foundation of China(No. 21974151).

Abstract: Three target peptides, H3K9ac, H3K14ac and H3, in human cell lines were determined by highperformance liquid chromatography coupled with triple quadrupole mass spectrometry under positive ion multiple reaction monitoring mode(MRM). The cell samples were pre-treated by trypsin digestion,propionylation derivatization, desalination, peptide enrichment, and then separated by ACQUITY UPLC BEH C₁₈ column. The results indicated that the three peptides in cells had good linear relationship within the concentration range of 1-250 ng/mL(R²>0.99). The detection limits(S/N=3) of the peptides were 0.1 ng/mL.Accuracy, precision, matrix effects and recoveries meet the methodological requirement for biological samples analysis. The established method had many advantages such as simple operation, high sensitivity,fast detection and accurate quantification, which had been initially applied to evaluate the effects of two classic genotoxic compounds(camptothecin and colchicine) with different modes of action on the acetylation at position 9 and position 14 of histone H3 in cells. This method provided important technical support for DNA damage/repair and transcription research of genotoxic compounds.

Key words:

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蛋白修饰定量质谱法研究两种不同作用模式基因毒化合物对组蛋白H3乙酰化的影响

通讯作者: 徐华,E-mail:huarxu@163.com

English

Determination of Genotoxic Compounds with Different Modes of Action on Histone H3 Acetylation by Protein Modification Quantitative Mass Spectrometry

Corresponding author: XU Hua, huarxu@163.com

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

蛋白修饰定量质谱法研究两种不同作用模式基因毒化合物对组蛋白H3乙酰化的影响

通讯作者: 徐华,E-mail:huarxu@163.com

English

Determination of Genotoxic Compounds with Different Modes of Action on Histone H3 Acetylation by Protein Modification Quantitative Mass Spectrometry

Corresponding author: XU Hua, huarxu@163.com

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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CCS Chemistry：颜徐州、鲍哲南：你的皮肤可能是一片SPMs